Bioactive molecules that are isolated from plants, bacteria, and fungi are often referred to as natural products. These molecules are synthesized by primary or secondary pathways within the organism or may even be degradation products of another molecule. However, many of these molecules have shown a variety of therapeutic uses in humans and other animal species. One of the best known examples is taxol, which was originally isolated from the bark of the Pacific Yew tree. Taxol has been shown to have anti-cancer properties and is currently used in the treatment of breast cancer.
Actinomycetes are prolific producers of bioactive small molecules, which may have a variety of clinical applications such as immunosuppressants, antibiotics, and cancer therapeutics. Actinomycetes are Gram-positive bacteria that form long, thread-like branched filaments. The term actinomycetes is used to indicate organisms belonging to the Actinomycetales, an Order of the domain Bacteria. The Actinomycetales are divided into 34 Families including Streptomyceteae, to which belongs the genus Streptomyces (Bergy's Manual of Systematic Bacteriology, Second Edition, 2001; George M. Garrity, Editor-in-Chief, Springer Verlag, New York).
The small molecules isolated from these organisms are produced by enzymes encoded within clusters of genes on the chromosomes of these organisms. In many cases the small molecules have a number of functional groups and complex stereochemistries which do not easily lend themselves to being reproduced synthetically. In order to make structural modifications to these molecules, changes in the biosynthetic genes responsible for their production are needed. However, vectors currently available for actinomycetes do not have the versatility of vectors used in E. coli, so DNA inserts have to be recloned in other vectors for purposes such as DNA sequencing or synthesis of RNA probes.
Shuttle vectors such as pFD666 (Denis & Brzezinski, Gene, 111:115, 1992) have been developed to speed up routine sub-cloning experiments. These vectors should facilitate the cloning, restriction mapping, DNA sequencing, and functional analysis of the actinomycetes genes. However, these vectors require transformation of E. coli, purification of the vector from the E. coli, and then transformation into actinomycetes. Transformation requires the development of protoplast formation and regeneration protocols each time it is necessary to introduce DNA into a new actinomycetes species, which decreases the simplicity of the process. Additionally, loss of vector and contamination may become factors. Therefore, alternative methods are needed.
An alternative method of performing this genetic manipulation is intergeneric conjugation, which utilizes a system of passing DNA from E. coli to actinomycetes directly, i.e., without isolation, purification and manipulation of the DNA by the investigator (Baltz et al., Trends Biotechnol. 1996; 14(7):245-50; Flett et al., FEMS Microbiol. Lett. 1997; 155(2):223-9; Mazodier et al., J. Bacteriol. 1989; 171(6):3583-5). Intergeneric conjugation has fewer manipulations than transformation, and therefore, the protocol development for each species is quicker. Additionally, the purification of significant quantities of plasmid DNA from E. coli is not required for intergeneric conjugation as it is in transformation.
There are a limited number of conjugation vectors that have been prepared to date, and many are derived from SCP2* origin of replication while other vectors have been prepared that comprise a pIJ101 origin of replication. (Keisser et al. Practical Streptomyces Genetics, John Innes Centre, Norwich, England, 2000). There is a continuing need in the art to develop bifunctional vectors that may be used to manipulate the biosynthetic genes responsible for small molecule formation.